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Reduced formation of depurinating estrogen-DNA adducts
by sulforaphane or KEAP1 disruption in human mammary epithelial MCF-10A cells
Journal: Carcinogenesis
Manuscript ID: CARCIN-2013-00184.R2
Manuscript Type: Original Manuscript
Date Submitted by the Author: 21-Jun-2013
Complete List of Authors: Yang, Li; University of Pittsburgh, Pharmacology & Chemical Biology
Zahid, Muhammad; University of Nebraska Medical Center, Environmental, Agricultural and Occupational Health Liao, Yong; University of Pittsburgh, Cell Biology Rogan, Eleanor; University of Nebraska Medical Center, Eppley Institute for Research in Cancer Cavalieri, Ercole; University of Nebraska Medical Center, Eppley Research Institute Davidson, Nancy; University of Pittsburgh Cancer Institute, ; Yager, James; Johns Hopkins University, Dept. of Environ. Health Sciences Visvanathan, Kala; Johns Hopkins, Epidemiology/Oncology Groopman, John; Johns Hopkins University, Bloomberg School of Public Health, Environmental Health Sciences;
Kensler, Thomas; Johns Hopkins School of Public Health, Environmental Health Sciences
Keywords: sulforaphane, estrogen-DNA adducts, Nrf2, Keap1, estrogen metabolism
Carcinogenesis Carcinogenesis Advance Access published July 10, 2013 at T
he Univesity of C
algary on July 29, 2013http://carcin.oxfordjournals.org/
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Reduced formation of depurinating estrogen-DNA adducts by sulforaphane or
KEAP1 disruption in human mammary epithelial MCF-10A cells
Li Yang1, Muhammad Zahid2, Yong Liao3, Eleanor G.Rogan2, Ercole L.Cavalieri4, Nancy
E.Davidson1,5, James D.Yager6, Kala Visvanathan7, John D.Groopman6, Thomas
W.Kensler1,5,6*
1 Department of Pharmacology & Chemical Biology, University of Pittsburgh School of
Medicine, Pittsburgh, PA, 15213
2 Environmental, Agricultural and Occupational Health, College of Public Health,
University of Nebraska Medical Center, Omaha, NE 68198
3 Department of Cell Biology, University of Pittsburgh School of Medicine
4 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska
Medical Center, Omaha, NE 68198
5 University of Pittsburgh Cancer Institute and UPMC Cancer Center
6 Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MD 21205
7 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD 21205.
*To whom correspondence should be addressed. Department of Pharmacology &
Chemical Biology, University of Pittsburgh, Room E1352, 200 Lothrop St, Pittsburgh, PA
15261, USA
Tel: +412-648-1942; Fax: +412-648-1945; Email: tkensler@pitt.edu
Running Title: Sulforaphane reduces estrogen-DNA adduct levels
Abbreviations: SFN, sulforaphane; E2, estradiol, E1, estrone; 4-OHE2, 4-
hydroxyestradiol; ER, estrogen receptor; NQO1, NAD(P)H:quinone oxidoreductase1;
GST, glutathione-S-transferases; COMT, catechol-O-methyltransferase; Keap1, Kelch-
like ECH-associated protein 1; Nrf2, NF-E2-related factor 2; UHPLC-MS/MS, ultra-
performance liquid chromatography - tandem mass spectrometry; CYP1A1, cytochrome
P450 1A1; CYP1B1, cytochrome P450 1B1.
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Abstract
Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as
NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GST) via the
Kelch-like ECH-associated protein 1 (Keap1) - NF-E2-related factor 2 (Nrf2) signaling
pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-
quinone, while GSTs detoxify it through conjugation with glutathione. These 3,4-
quinones can react with DNA to form depurinating DNA adducts. Thus, SFN may alter
estrogen metabolism and thus protect against estrogen-mediated DNA damage and
carcinogenesis. Human breast epithelial MCF10A cells were treated with either vehicle
or SFN and either estradiol (E2) or its metabolite 4-OHE2. 4-Hydroxy derived estrogen
metabolites and depurinating DNA adducts formed from E2 and its interconvertable
metabolite estrone (E1) were analyzed by mass spectrometry. Levels of the depurinated
adducts, 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-N7Guanine, were reduced by 60% in
SFN treated cells, while levels of 4-OCH3E1/2 and 4-OHE1/2- glutathione conjugates
increased. To constitutively enhance the expression of Nrf2-regulated genes, cells were
treated with either scrambled or siKEAP1 RNA. Following E2 or 4-OHE2 treatments,
levels of the adenine and guanine adducts dropped 60% - 70% in siKEAP1 treated cells
while 4-OHE1/2-glutathione conjugates increased. However, 4-OCH3E1/2 decreased 50%
after siKEAP1 treatment. Thus, treatment with SFN or siKEAP1 has similar effects on
reduction of depurinating estrogen-DNA adduct levels following estrogen challenge.
However, these pharmacologic and genetic approaches have different effects on
estrogen metabolism to O-methyl and glutathione conjugates. Activation of the Nrf2
pathway, especially elevated NQO1, may account for some but not all of the protective
effects of SFN against estrogen-mediated DNA damage.
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Introduction
Elevated levels of estrogens have been recognized as an important determinant of
the risk of breast cancer [1]. Studies in experimental animal models demonstrate that
estradiol (E2) and estrone (E1) are carcinogenic [2] and studies in cultured human cells
[3; 4] provide a mechanistic basis for this effect. Observational studies and clinical trials
consistently support the contention that sustained exposure to endogenous estrogens is
associated with the development of sporadic breast cancer. Two complementary
pathways are likely required for estrogen carcinogenicity [2]. One involves signaling
through the estrogen receptor (ER) leading to altered gene expression and increased
proliferation accompanied by spontaneous mutations [5]. The other pathway, outlined in
Figure 1, involves the oxidative metabolism of E1 or E2 to catechol estrogens and then
reactive quinone metabolites. These metabolites can then directly and/or indirectly
cause DNA damage and mutations responsible for the initiation and progression to
breast cancer.
Metabolism of estrogens is characterized by a balanced set of activating and
deactivating pathways. Aromatization of androstenedione and testosterone by
aromatase (CYP19) yields E1 and E2, respectively. E1 and E2 are interconverted by 17β-
hydroxysteroid dehydrogenase, and they are metabolized at the 2- or 4-position to form
2-OHE1/2 or 4-OHE1/2, respectively. CYP1A1 preferentially hydroxylates E1 and E2 at C-2,
whereas CYP1B1 almost exclusively catalyzes the formation of 4-OHE1/2 [6]. The most
common pathway of conjugation of estrogens in extrahepatic tissues is O-methylation,
catalyzed by catechol-O-methyltransferase (COMT). If the activity of COMT is low, CYP
or peroxidases can catalyze competitive oxidation of the catechol estrogens to E1/2-2,3-
quinones and E1/2-3,4-quinones. Higher levels of depurinating DNA adducts are formed
by E1/2-3,4-quinones compared to E1/2-2,3-quinones due to different mechanisms of
adduction [7]. The E1/2-3,4-quinones react via a 1,4-Michael addition, whereas the E1/2-
2,3-quinones rearrange to p-quinone methides, which react via a 1,6-Michael addition
[8]. These adducts generate apurinic sites that can be converted into mutations by error-
prone repair, which in turn may initiate breast carcinogenesis [9]. Consistent with these
actions, E1/2-3,4-quinone produced A-T to G-C mutations in the DNA of the mammary
gland of ACI rats [10].
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While the estrogen oxidation pathway is detrimental to the integrity of DNA, several
protective pathways in cells control the homeostasis of estrogen metabolism to avoid
DNA damage. Catechol estrogens can be detoxified by COMT, and the E1/2-3, 4-
quinones by conjugation with glutathione or by reduction back to catechol estrogens,
catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) [8] (Fig.1). Diminished
expression of detoxication enzymes and/or upregulation of enzymes of the oxidation
pathway can disrupt this homeostasis. For example, higher expression of CYP19 and
CYP1B1, or lower expression of COMT and NQO1, in breast tissues is associated with
elevated risk of breast cancer [11]. Gaikwad et al. [12; 13] and Pruthi et al. [14] have
reported that there is a significantly higher ratio of depurinating DNA adducts to other
estrogen metabolites when comparing women at high risk for breast cancer or
diagnosed with the disease to controls, indicating that formation of depurinating
estrogen-DNA adducts likely plays key roles in breast cancer development.
Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with
particularly high levels in 3-day old broccoli sprouts (15). It is converted by hydrolysis of
the glucosinolate, glucoraphanin, by the enzyme, myrosinase, found in plants or by β-
thioglucosidases found in the gut microflora. SFN is an attractive chemopreventive agent
since it is safe and can be distributed widely as broccoli sprout preparations. Moreover,
SFN and broccoli sprout preparations are effective chemopreventive agents in rodent
models of mammary carcinogenesis (15; 16) and initial pharmacokinetic studies indicate
that pharmacologically relevant concentrations of sulforaphane metabolites can be
detected in the mammary epithelium of women consuming broccoli sprout derived
beverages (17). An important, but far from unilateral, mechanism of action for SFN is the
induction of carcinogen detoxication enzymes such as NQO1 and glutathione S-
transferases (GSTs). SFN is an activator of the antioxidant response element -Keap1-
Nrf2 signaling pathway regulating the expression of these and many other genes [18].
Under normal cellular conditions, Nrf2 binds to Keap1 in the cytoplasm, resulting in
ubiquitination of Nrf2 and its subsequent proteasomal degradation [19]. SFN can modify
cysteine 151 in Keap1 to disrupt the association of Cul3 ubiquitin ligase with Keap1,
allowing Nrf2 to escape degradation. Thus, Nrf2 is stabilized and translocates into the
nucleus to induce the transcription of its target genes such as NQO1 and GST [3,20].
Using transcriptomic and proteomic profiling, we have shown previously, that SFN
induces Nrf2-regulated genes in ERα negative, non-tumorigenic human breast epithelial
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MCF-10A and MCF-12A cells (20), and primary cultures of human mammary epithelial
cells (21). Interesting, these profiles were similar to those provoked by treatment of the
MCF-10A cells with siRNA vectors for KEAP1 knockdown (20). In the present study, we
have evaluated the efficacy of SFN to alter estrogen metabolism away from the
formation of DNA adducts through induction of detoxication genes and to determine the
extent to which such protection can be mimicked by genetic amplification of the Nrf2
signaling pathway through KEAP1 knockdown.
Materials and Methods
Chemicals and reagents
E2, 4-OHE2 and 4-OCH3E1 (E2) were obtained from Steraloids, Inc. (Newport, RI).
Standards of the glutathione conjugates of 4-OHE2 and the depurinating DNA adducts
were synthesized by published procedures [7, 22-24]. A mixed standard solution of 10
µg/mL was obtained by mixing 10 µL of individual stock solutions (1 mg/mL) and diluting
to 1 mL with methanol/water 50:50 with 0.1% formic acid. Serial dilutions of this solution
were used as standards for the preparation of calibration curves. Stock standard
solutions and mixed standard solutions were stored at −80 ºC. Sulforaphane (1-
isothiocyanato-4-(methylsulfinyl)-butane) was purchased from LKT Laboratories (St.
Paul, MN). The glutathione (reduced) quantification kit was purchased from Dojindo
Molecular Technologies (Rockville, MD). All other chemicals were the highest quality
obtainable commercially.
Cell lines and cell culture
MCF-10A cells were obtained from the American Type Culture Collection and
cultured in estrogen-free medium at 37ºC in a humidified incubator containing 5% CO2.
Cell culture medium was prepared by a phenol red-free mixture of Dulbecco’s modified
Eagle’s media and Ham’s nutrient mixture F-12 media (Mediatech, Manassas, VA)
containing 20 ng/mL epidermal growth factor, 10 µg/mL insulin, 0.5 µg/mL
hydrocortisone, 100 ng/mL cholera toxin and 5% charcoal-stripped FBS (Invitrogen, Inc.)
(25).
For SFN treatment, cells were seeded in 10-cm dishes (1–2×106 cells/10 ml
media/dish) overnight and then treated with either vehicle or SFN for 24 hr. After
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discarding the media, all plates were fed with fresh cell culture media containing 10 µM
E2 or 4-OHE2 for 24 hr and then re-treated with vehicle or SFN for another 24 hr (Figure
2) without changing cell culture media. SFN and E2 were dissolved in DMSO and 4-
OHE2 was dissolved in ethanol; the final vehicle concentration in the media was < 0.05%
(v/v). For siRNA knock down of KEAP1, cells were seeded in 10-cm dishes (1–2×106
cells/10 ml medium/dish) overnight and were transfected at 30-40% confluence with
siRNA human KEAP1(J-012453-07) or scrambled siRNA control(D-001810-01) from
Thermo Scientific (Dharmacon, Lafayette, CO) with lipofectamine 2000 for 48 hr
following the manufacturer's protocol (Figure 2). Confirmatory experiments were
conducted using a shRNA construct for KEAP1. The target sequence selected, 5′-
GTGGGCGTGGCTGTCCTCAAT -3′ (sense), corresponds to a region 1572 to 1592 bp
of the Keap1 gene. The sh-Scramble sequence was selected as 5′-
GGACGGAGCAGTCAAGTACAA-3′. The targeted and scrambled sequences were
subcloned into entry vector pENTR/H1 plasmid (Invitrogen), pENTR/H1/Keap1 or
scramble was recombined into pAD/Dest. Then pAd/Keap1 or scramble was transfected
into the adenovirus packaging cell line. After generation, amplification and titer
measuring, the adenoviruses were then incubated with MCF-10A cells at 37°C. The cell
culture media were collected with 2 mg/mL ascorbic acid added and either processed
immediately or frozen at -80ºC prior to assay for estrogen metabolites and depurinating
DNA adducts. Cells were harvested for RNA, protein and activity assays.
qRT-PCR
Total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen,
Germantown, MD) or 5 PRIME PerfectPure RNA Cell & Tissue Kit (5PRIME,
Gaithersburg,MD), from which cDNA was synthesized using the qScriptTM cDNA
synthesis kit (Quanta BioSciences, Gaithersburg, MD). PCR was carried out in a 20 µl
volume including each target primer, cDNA and iQ™ SYBR® Green Supermix (Bio-Rad,
Hercules, CA) and run in a Bio-Rad Thermal Cycler (Bio-Rad, Hamburg, Germany).
Fold-change values were determined using the 2-∆∆Ct relative quantification method [26].
The amplified products were electrophoresed on agarose gel and stained with ethidium
bromide.
PCR primers were as follows: forward 5′-TGACAATGAGGTTTCTTCGG-3′
and reverse 5′-TCTGTCAGTTTGGCTTCTGG-3′ for human Nrf2; forward 5′-
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ACGTCCTTGGAGGCTATGAT-3′ and reverse 5′-TCTGCTGGTCAATCTGCTTC -3
′ for human KEAP1; forward 5′- CGCTTCTCTTGGAGGAATGT -3′ and reverse 5′
- TCCACCACCTCCCTGTATTC -3′ for human COMT; forward 5′-
TTCCGGAGTAAGAAGGCAGT -3′ and reverse 5′- GGAGTGTGCCCAATGCTAT -3
′ for human NQO1; forward 5′- TAAAGGAGAGAGCCCTGATTG -3′ and reverse 5
′- TTCAAAGGCAGGGAAGTAGC -3′ for human GSTA1; forward 5′-
GGACTCATGACCACAGTCCA -3′ and reverse 5′- CTGCTTCACCACCTTCTTGA -3
′ for GAPDH.
Western blots
After treatment, cells were harvested and then lysed in RIPA buffer with protease
inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) and unlysed cellular debris
removed by centrifugation. Protein concentrations were determined by using the BCA
protein assay kit (Pierce Biotechnology, Rockford, IL). Western blot procedures followed
the ABC protocol. Samples were electrophoresed on SDS-PAGE and transferred to
polyvinylidene difluoride membranes. The membranes were blocked in TBS-0.05%
Tween 20 with 5% non-fat milk, incubated with primary antibodies, and incubated with a
peroxidase-conjugated secondary antibody after extensive washing. Dilutions of primary
anti-KEAP1, Nrf2, NQO1 (Santa Cruz), CYP1B1 (Genetest, Bedford, A), COMT, GSTA1
and β-actin (Sigma) antibodies, were made in blocking solution (5% non-fat dry milk in
TBS). The blots were incubated with Western Lightning® Plus-ECL solution
(PerkinElmer, Waltham, MA) and visualized with X-ray film. Intensities of the bands were
quantified by Bio-Rad Quantity One® software (Bio-Rad, Hercules, CA). The
densitometry ratios for treated samples compared to controls were determined for three
biological replicates and normalization was to β-actin.
NQO1 activity
MCF-10A cells were washed three times with 0.25 M sucrose/10 mM potassium
phosphate (pH 7.2),collected from the plates by scraping, frozen in liquid nitrogen, and
stored at -80°C for assay as described previously (27).
UPLC-MS/MS analysis of estrogen metabolites and depurinating DNA adducts
Extraction of estrogen metabolites and depurinating DNA adducts from cell
culture media was modified from previously described procedures (12). After adjusting
the pH to 7, cell culture media (10 mL/sample) was loaded onto phenyl cartridges
(Agilent Technologies) that were preconditioned with methanol and water. Extracts were
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eluted as described (12), lyophilized, re-dissolved in a methanol: water 50:50 mixture
containing 0.1% formic acid, and finally subjected to UPLC-MS/MS analysis. Mass
spectrometer parameters are presented in Table 1. Analyses were conducted using
selected reaction monitoring with a triple stage quadrupole mass spectrometer (TSQ
Vantage, Thermo Scientific, USA) by using heated electrospray ionization in positive ion
mode. The mass spectrometer was interfaced to a UPLC system consisting of an Accela
quaternary pump (Thermo, USA) used for the chromatographic separation, and a
Thermo Pal autosampler (HTC PAL, Zwingen, Switzerland). A Hypersil Gold column (1.9
µm, 100×2.1 mm, Thermo Electron) was used for separation at a flow rate of 0.5
mL/min. The gradient started with 95% A (0.1% formic acid in H2O) and 5% B (0.1%
formic acid in CH3CN), changed to 80% A over 1 min, changed to 79% A over 4 min,
followed by a 4-min linear gradient to 30% A, changed to 2% A for 2 minutes, then
changed back to the original conditions with a 3-minute hold, resulting in a total
separation time of 14 min. For all the studies, a methanol: water (1:1) mixture with 0.1%
formic acid was used as the carrier solution. A signal-to-noise ratio of 3 was used as the
limit of detection for each compound. Experiments were performed by applying a
capillary (ion transfer tube) temperature of 380 ºC, vaporizer temperature 398 ºC, sheath
gas pressure (arbitrary units) 50, auxiliary gas pressure (arbitrary units) 20, spray
voltage 3.98kV, collision gas pressure 1.5 mTorr. The collision energy for each
compound is listed in Table 1. The coefficient of variance for all analytes was < 4%.The
Xcalibur software (Thermo) was used to process and quantify the acquired data of
estrogen metabolites.
Statistics
The data are presented as means ± standard error of at least three independent
experiments. Comparisons between two groups were analyzed using the Student t-test,
and significance was established at p < 0.05 using Prizm 5 software.
Results
Modulation of transcripts, protein expression and activity of estrogen metabolism
enzymes by SFN treatment or KEAP1 knockdown
Treatment of MCF-10A cells with SFN led to induction of GSTA1 and NQO1
transcripts but no changes in expression levels of two other genes known to influence E2
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metabolism, namely CYP1B1 and COMT, were observed (Fig. 3A). These inductions
exhibited a dose-response, with minimal induction observed with 3 µM, moderate with 7
µM and near maximal with 10 µM SFN (data not shown). As expected, there were also
no changes in the transcript levels of KEAP1 or NRF2. SFN treatment significantly
elevated NQO1 protein level 3.0 fold (p<0.01; SFN treatment versus vehicle, Fig. 3B)
and its specific activity 2.7-fold (p<0.01; SFN treatment versus vehicle, Fig. 3C).
Although no change of COMT mRNA level was detected, a significant 2.4-fold increase
in COMT protein (p<0.05; SFN treatment versus vehicle, Fig. 3B) was observed.
CYP1B1 protein was significantly decreased by 50% with SFN treatment (Fig.3B)
(p<0.05; SFN treatment versus vehicle). Thus, it appears SFN influences the expression
of E2 metabolizing enzymes through both transcriptional and post-transcriptional
mechanisms. As expected, SFN and siKEAP1 also lead to increases in intracellular
concentrations of reduced glutathione in MCF-10A cells (1.48 and 1.71-fold,
respectively).
Since SFN is a well characterized activator of Nrf2 signaling in MCF-10A and
other mammary cells (20,21), the influence of siKEAP1 knockdown, and hence genetic
activation of the pathway was evaluated. Shown in Fig. 4, transcript (Fig. 4A), protein
(Fig. 4B) and specific activity of NQO1 (Fig. 4C) significantly increased in the setting
where KEAP1 expression was significantly reduced by 80% (p<0.01 siKEAP1 versus
scrambled). Interestingly, no induction of GSTA1 transcripts was detected, suggesting
that the SFN-mediated induction of this gene is Nrf2-independent. Also unexpectedly,
levels of COMT transcripts as well as COMT protein were significantly decreased 60-
70% by the siKEAP1 treatment (p<0.01 for COMT protein level in siKEAP1 versus
scrambled) (Fig 4A & 4B). Comparable results were seen using shKEAP1 knockdown
(data not shown). COMT is not known to be a direct Nrf2 regulated gene and the
mechanism underlying this response is not known.
Modulation of levels of depurinating estrogen-DNA adducts and estrogen
metabolites by SFN or siKEAP1 treatment
At 48 hr after E2 treatment, the summed levels of the depurinating adducts, 4-
OHE1/2-1-N3Adenine and 4-OHE 1/2-1-N7Guanine, in the culture media were significantly
lower in SFN treated cells compared to vehicle (0.03 ± 0.01 versus 0.07 ± 0.02
pmole/106cell, p<0.05) (Fig. 5A). Although E2 was added to the cells, there was
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considerable conversion to E1. Approximately half of the formed adenine and guanine
adducts were derivatives of E1 and the remainder were from E2. By contrast, levels of 4-
OCH3E1/2 increased significantly with SFN treatment (5.36 ± 0.16 versus 1.81 ± 0.20
pmole/106cell, p<0.01) (Fig. 5B). More modest increases in the levels of 4-OHE1/2-
glutathione conjugates were measured following SFN treatment (1.54 ± 0.37 versus 0.83
± 0.19 pmole/106cell, p<0.05, Fig. 5C). About 25-fold more methoxy conjugates were
formed than glutathione conjugates in either the vehicle or SFN treated cells. 2-OHE1/2
adducts were also measured, but typically reflected only 2-3% of the level of the 4-
OHE1/2 DNA adducts formed. Therefore, we did not characterize patterns of 2-OHE1/2-
derived metabolites.
Addition of the proximate metabolite, 4-OHE2, to cells led to 20-fold higher levels
of depurinating estrogen-DNA adducts than seen with E2 in vehicle treated cells. In this
instance the majority were derived from E1. 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-
N7Guanine adducts were again significantly lower in SFN treated cells compared to
vehicle (0.59 ± 0.11 versus 1.42 ± 0.16 pmole/106cell, p<0.01, Fig 5D). 4-OCH3E1/2
levels increased 3.4-fold (195.00 ± 12.33 versus 58.05 ± 1.77 pmole/106cell, p<0.01, Fig.
5E) while 4-OHE1/2-glutathione-conjugates increased 5.1-fold following SFN treatment
(4.44 ± 0.52 versus 0.87 ± 0.03 pmole/106cell, p<0.01, Fig. 5F). The methoxy conjugates
were the dominant metabolites detected.
Pretreatment of cells with siKEAP1 led to more substantial declines in levels of
the depurinating estrogen-DNA adducts in the cell culture media. Following E2 treatment,
levels of the 4-OHE1/2-1-N3Adenine and 4-OHE1/2-1-N7Guanine adducts dropped 70% in
siKEAP1 treated cells compared to scrambled vector. 4-OCH3E1/2 levels decreased 50%
with siKEAP1 treatment (Fig. 5E), while levels of the 4-OHE1/2-glutathione conjugates
were not significantly different (Fig. 5F). Both outcomes are consistent with the effects of
siKEAP1 on COMT and GSTA1 transcripts (Fig. 4A). Similar results were seen following
4-OHE2 treatment. 4-OHE1/2-1-N3Adenine and 4-OHE 1/2-1-N7Guanine adducts declined
90% in siKEAP1 treated cells compared to scrambled vector (Fig. 5D); 4-OCH3E1/2
declined 60% (Fig. 5E) while there was no significant change in levels of 4-OHE1/2-
glutathione conjugates with siKEAP1 treatment. (Fig. 5F)
Discussion
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The natural and synthetic estrogens E1, E2, diethylstibestrol and hexestrol induce
kidney tumors in Syrian golden hamsters [28]. Additionally, the proximate estrogen
metabolites, 4-OHE1/2, are carcinogenic in hamsters and mice, but 2-OHE1/2 metabolites
are not [29-31]. 4-OHE2 and E2-3,4-quinone are mutagenic in mouse skin and rat
mammary gland [9,32]. The genotoxicity of 4-OHE2 and E2-3,4-quinone was also
demonstrated in the Big Blue rat embryonic cell line, but no mutations were observed
after treatment of the cells with 2-OHE2 [33]. The much greater carcinogenic activity of 4-
OHE1/2 compared to 2-OHE1/2 likely reflects the far greater propensity of E1/2-3,4-
quinones to form estrogen-DNA adducts compared to E1/2-2,3-quinones [7]. Transgenic
mice with ERα knocked out (ERKO/wnt-1 mice) provide another model that
demonstrates the critical role of estrogen genotoxicity in carcinogenesis. The wnt-1
transgene induced mammary tumors in female ERKO/wnt-1 mice, despite the lack of
ERα. [34]. Mammary tumors developed in these mice even when ovariectomized mice
were implanted with both E2 and the anti-estrogen ICI-182,780 [35] suggesting that non-
ER pathways such as metabolism to the genotoxic quinones that form the adenine and
guanine adducts contribute to mammary tumor development.
MCF-10A cells are an immortalized mammary epithelial cell line lacking ERα.
Treatment of MCF-10A cells with E2 or 4-OHE2 generates the depurinating estrogen-
DNA adducts and transformation of the cells, as detected by their ability to form colonies
in soft agar [36]. This transformation can also occur in the presence of the antiestrogens,
tamoxifen or ICI-182,780 [37]. Thus, DNA damage and cell transformation by estrogens
do not require a functional ERα signaling pathway. Consequently, strategies to prevent
the formation of estrogen-DNA adducts may alter the initiation and development of both
ERα positive and negative cancers.
The depurinating estrogen-DNA adducts that efflux from cells and tissues are
excreted in urine, allowing their identification and quantification as biomarkers of risk of
developing breast cancer [7, 12-14]. High levels of estrogen-DNA adducts have been
observed in analyses of urine and serum from women at high risk for or diagnosed with
breast cancer compared to women at normal risk (12-14). Levels of DNA adducts are
strongly influenced by the balance of enzymes involved in their bioactivation to reactive
intermediates and their detoxication. Protective enzymes such as COMT, GSTs, and
NQO1 can decrease steady-state levels of E1/2-3-4-quinones and the resulting
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depurinating estrogen-DNA adducts in cell culture models (Fig. 1). For example,
inhibition of COMT led to an increase in oxidative DNA damage in human breast cancer-
derived MCF-7 cells, [38] and formation of E1/2-3-4-quinone adenine and guanine DNA
adducts in MCF-10F cells [39]. Conjugation of the quinones with glutathione catalyzed
by GSTs and reduction by NQO1 of the quinones back to catechol estrogens can be
envisioned to mitigate the formation of depurinating adducts. A series of isogenic MCF-
10F cells stably expressing polymorphic variants of NQO1 with lower capacity to reduce
estrogen quinones compared to wild-type NQO1 consistently led to increased formation
of estrogen-DNA adducts from E2-3,4-quinone (40).
As seen in an earlier study using the phytochemical resveratrol in MCF-10F cells
it is possible to reduce the formation of estrogen-DNA adducts and to decrease E2-
induced transformation to anchorage-independent growth [4]. We show here that the
broadly effective chemopreventive agent isolated from broccoli, SFN, also exerts strong
protective effects against the DNA damaging actions of estrogens in the MCF-10A cells.
The enzymes associated with protection are inducible by SFN in many human cell types,
including MCF-12A (20) and primary mammary epithelial cells (21). Given the current
pre-clinical and clinical evaluation of SFN (typically as an enriched component of
broccoli sprout extracts) (see ClinicalTrials.gov), there is interest in targeting this agent
towards breast cancer prevention. Protection against the genotoxicity of estrogens in the
MCF-10A cells can be induced by treatment with SFN or genetic activation of Nrf2
signaling. However, the underlying metabolic changes are distinct, indicating that
upregulation of Nrf2 signaling may not account for the full effects of SFN on this
endpoint. Following treatment with E2, the SFN pre-treated cells exhibit significant
increases in the production of 4-OCH3 and glutathione conjugates. However, these
detoxication metabolites were not elevated by the pretreatment of cells with siKEAP1.
Thus, the effects of SFN on COMT and GSTA1 appear to occur independently of Nrf2
signaling. Indeed, siKEAP1 led to a decrease in COMT protein levels and decline in 4-
OCH3 formation while nonetheless exhibiting protection. Whether Nrf2 exerts negative
regulation on COMT is not known although neither microarray nor ChIP-Seq studies
indicate it to be a direct Nrf2 target gene (41; 42). Presumably then, SFN-mediated up-
regulation of COMT levels at the protein, but not transcript level, reflects a distinct, Nrf2-
independent mode of action. Given the protective actions of both the pharmacologic and
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genetic interventions, and the common induction of NQO1 by both modalities, it is likely
that reduction of the E1/2-3,4-quinones back to catechol estrogens is central to the effect.
SFN also reduces levels of CYP1B1 protein in these cells, which could dampen
formation of the catechol estrogens. However, the protection by SFN is also seen when
4-OHE2 is administered to cells, a metabolite that is a product of CYP1B1 action. Thus, it
is unlikely that inhibition of estrogen bioactivation is critical to protection in this setting.
That bioactivation of E2 is critical to its genotoxicity is underscored by the observation
that 20-fold higher levels of depurinating estrogen-DNA adducts are detected following
administration of 4-OHE2 than with E2. It should also be noted that considerably more
catechol-O-methyl conjugates are formed than glutathione conjugates (~200 pmol/106
cells versus 4 pmol/106 cells), indicating that GST catalyzed detoxication plays a minor
role in overall protection against estrogen genotoxicity. Moreover, the absence of
induction of GSTA1 by siKEAP1, which is nonetheless protective, also signals that GSTs
are not critical in modulating estrogen-DNA adduct burden. Elevation of intracellular
glutathione, observed in both the pharmacologic and genetic manipulations, did not lead
to elevated 4-OH-estrogen-glutathione conjugates in the siKEAP1-treated cells,
suggesting that non-enzymatic conjugation with glutathione too has no protective role.
In conclusion, SFN is the embodiment of phytochemical poly-pharmacy in a
single molecule. It interacts with many molecular targets in cells and exerts its
chemopreventive actions through actions on multiple pathways (43). The protective
effect of SFN against estrogen mediated DNA damage further highlights its possible role
in chemoprevention of mammary carcinogenesis and illustrates that multiple
mechanisms likely account for this outcome. Induction of the Nrf2-regulated detoxication
gene, NQO1, would seem to be central to the protective alterations in metabolite
distribution. At the same time, Nrf2-independent actions of SFN on COMT and GSTA1
are likely to contribute to enhanced protection of the genome.
Funding: This work was supported by DOD BCRP Postdoctoral Fellowship103928,
USPHS Breast SPORE P50 CA088843, and a Pennsylvania Department of Health
Commonwealth Universal Research Enhancement Grant.
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Figure Legends
Figure 1. Pathway for formation of estrogen depurinating DNA adducts. Estradiol (E2) or
estrone (E1) can be oxidized to E1/2 -3,4-quinone, which can bind to DNA to form 4-
OHE1/2-1-N3Adenine or 4-OHE1/2-1- N7Guanine adducts. NQO1 reduces E1/2 - 3, 4-
quinones back to catechols and GST catalyzes the conjugation of E1/2 -3,4-quinones with
glutathione while COMT catalyzes the methylation of 4-OHE1/2 to 4-OCH3E1/2.
Figure 2. Timeline for treatments of MCF-10A cells
Figure 3. Effects of sulforaphane on transcript, protein and activities of enzymes
metabolizing estradiol or estrone. A. Effect of SFN on transcripts levels of estrogen
metabolism enzymes. B. Effect of SFN on protein levels of estrogen metabolism
enzymes. C. Effect of SFN on NQO1 activity. MCF-10A cells were treated with 10 µM
SFN as described in Figure 2. Values are mean ± SE of 3 independent experiments. *,
Differs from vehicle control, p < 0.05.
Figure 4. Effects of siKEAP1 on transcript, protein and activities of estradiol
metabolizing enzymes. A. Effect of siKEAP1on levels of estrogen metabolism enzyme
transcripts. B. Effect of siKEAP1on protein levels of estrogen metabolism enzymes. Scr,
scrambled; siKp, siKEAP1. C. Effect of siKEAP1 on NQO1 activity. MCF-10A cells were
treated with siKEAP1 as described in Figure 2. Values are mean ± SE of 3 independent
experiments. *, Differs from scrambled control, p < 0.05.
Figure 5. Effect of pharmacologic or genetic perturbation of estrogen metabolism on
estrogen-DNA adducts and metabolites in MCF-10A cells. Cell culture media were
collected, partially purified by solid phase extraction, and analytes separated and
quantified by UPLC-/MS/MS. A, B, C. Levels of estrogen-DNA adducts, 4-OHCH3E1/2 or
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E1/2 -glutathione conjugates, respectively, following addition of E2 to cells pre-treated with
either SFN or siKEAP1. D, E, F. Levels of estrogen-DNA adducts, 4-OHCH3E1/2 or E1/2 -
glutathione conjugates, respectively, following addition of 4-OHE2 to cells pre-treated
with either SFN or siKEAP1. Veh, vehicle; Scr, scrambled vector. Values are mean of
triplicate biological replicates ± SE. * Differs from control, p<0.05. **, Differs from vehicle
control, p < 0.01.
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Table1. Mass Spectrometry Parameters Name Parent Product Collision
Energy LOD (fmole)
4-OCH3E1 301.18 189.12 21 166.0 4-OCH3E2 303.15 137.10 23 329.9 4-OHE1-2-glutathione 592.16 317.06 23 84.4 4-OHE2-2-glutathione 594.17 319.08 24 168.3 4-OHE1-1-N7Guanine 436.20 152.02 39 2.8 4-OHE2-1-N7Guanine 438.21 272.12 35 2.79 4-OHE1-1-N3Adenine 420.20 296.09 44 2.91 4-OHE2-1-N3Adenine 422.16 136.00 47 2.9 2-OHE1-1-N3Adenine 420.20 136.04 31 1.43 2-OHE2-1-N3Adenine 422.22 136.02 28 1.42
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245x93mm (96 x 96 DPI)
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279x361mm (300 x 300 DPI)
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